induction of dnmt3b by pge2 and il6 at distant metastatic ... · induction of dnmt3b by pge2 and...

Induction of DNMT3B by PGE2 and IL6 at distant metastatic sites promotes

epigenetic modification and breast cancer colonization

Jae Young So,1 Nicolas Skrypek,

1 Howard H. Yang,

1 Anand S. Merchant,

2 George W. Nelson,

2

Wei-Dong Chen,1,3

Hiroki Ishii,1 Jennifer M. Chen,

1 Gangqing Hu,

4 Bhagelu R Achyut,

1,5 Esther

C. Yoon,6 Liying Han,

6 Chuanshu Huang,

7 Margaret C. Cam,

2 Keji Zhao,

4 Maxwell P. Lee,

1 and

Li Yang1,*

1Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer

Institute, National Institutes of Health, Bethesda, MD 20892 2Collaborative Bioinformatics Resource, Center for Cancer Research, National Cancer Institute,

National Institutes of Health, Bethesda, MD 20892 3Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of

Health, Bethesda, MD 20892 4Systems Biology Center, National Heart, Lung, and Blood Institute, National Institutes of

Health, Bethesda, MD 20892 5current address: Georgia Cancer Center, Augusta University, Augusta, GA 30912

6Department of Pathology, New York Medical College, Valhalla, NY 10595

7Department of Environmental Medicine and Biochemistry and Molecular Pharmacology, New

York University School of Medicine, Tuxedo, NY, 10987

Running title:

Inflammation-induced DNMT3B promotes metastasis

Keywords:

Metastasis, Epigenetic regulation, DNMT3B, PGE2, IL6

Conflict of interest statement:

The authors declare no potential conflicts of interest

*Correspondence:

Li Yang

[email protected]

Building 37, Room 3134C 37 Convent Drive, Bethesda, MD 20892

Tel: 240-760-6809

FAX: 301-402-1031

on April 20, 2020. © 2020 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 7, 2020; DOI: 10.1158/0008-5472.CAN-19-3339

mailto:[email protected]

http://cancerres.aacrjournals.org/

2

Abstract

Current cancer treatments are largely based on the genetic characterization of primary tumors

and are ineffective for metastatic disease. Here we report that DNA methyltransferase 3B

(DNMT3B) is induced at distant metastatic sites and mediates epigenetic reprogramming of

metastatic tumor cells. Multi-omics analysis and spontaneous metastatic mouse models revealed

that DNMT3B alters multiple pathways including STAT3, NFκB, PI3K/Akt, β-catenin, and

Notch signaling, which are critical for cancer cell survival, apoptosis, proliferation, invasion, and

colonization. PGE2 and IL-6 were identified as critical inflammatory mediators in DNMT3B

induction. DNMT3B expression levels positively correlated with human metastatic progression.

Targeting IL-6 or COX-2 reduced DNMT3B induction and improved chemo- or PD1- therapy.

We propose a novel mechanism linking the metastatic microenvironment with epigenetic

alterations that occur at distant sites. These results caution against the "Achilles' heel" in cancer

therapies based on primary tumor characterization and suggests targeting DNMT3B induction as

new option for treating metastatic disease.




3

Statement of Significance

Findings reveal that DNMT3B epigenetically regulates multiple pro-oncogenic signaling

pathways via the inflammatory microenvironment at distant sites, cautioning the clinical

approach basing current therapies on genetic characterization of primary tumors.




4

Introduction

Metastasis accounts for 90% of cancer mortality. Current therapies are largely based on

genotype variations and oncogene activation in primary tumor biopsies, however are ineffective

in treating metastatic diseases (1). Conventional chemotherapies and targeted therapies, which

are widely used in standard care, have a negative impact on patient quality of life and may

induce additional mutations that foster cancer and metastasis (1). Low response rate, therapy

relapse and resistance are challenges in immunotherapies that have yet to be overcome, despite

recent progress and optimism (2,3). In the metastatic process, tumor cells undergo invasion,

intravasation, extravasation, as well as survival and proliferation in the foreign organ

microenvironment to form clinically overt metastases. In particular, metastatic colonization at the

distant organ is a time-limiting step. Understanding the mechanism and adaptation of metastatic

tumor cells and their interaction with distant-organ microenvironment should uncover novel

therapeutic targets and rational combination treatment.

Tumor cells acquire metastatic capacity through several mechanisms. In the Darwinian-

like somatic evolution hypothesis, the metastatic clones and subclones in the primary tumor

continue to evolve during progression and treatment, resulting in substantial genetic divergence,

such as mutations in the estrogen receptor ligand-binding domain (4). In addition, acquired

driver mutations have been found in distant metastases that are not seen in the primary tumor in

breast cancer patients as well as in treatment-naive pancreatic cancer patients (5). Indeed,

significant genomic heterogeneity was observed in disseminated cancer cells after “curative”

surgery (6-8). Genomic alterations were also found in the overt metastasis of breast cancer when

compared with disseminated tumor cells (9,10). These studies indicate that the acquisition of

genetic changes can occur outside of the primary tumors in the distant organ sites.




5

Epigenetic alterations are another important mechanism for cancer cells to acquire key

traits of full malignancy (11). DNA methylation is one of the most consistent epigenetic changes

in human cancers (12). In fact, DNA methylation pattern is one of the strongest molecular

classifications in clustering cells of tumorigenic origin in the Pan-Cancer Atlas (13) and is

associated with metastatic risk (14-16). Of the three major DNA methyltransferases, DNMT3B

mediates de novo gene methylation and cooperates with DNMT1 to silence genes (17).

DNMT3B is upregulated in a number of human cancers and is associated with poor prognosis in

human breast cancers (18,19). DNA methylation is an epigenetic mechanism that promotes

cancer cell survival (20) and could be induced by non-genetic stimuli, such as oxidative stress

and pro-inflammatory molecules (21-23). However, whether regulation of metastatic tumor

methylomes occurs at the distant sites and if so, what are the underlying molecular mechanisms

and biological consequences remain unknown.

The premetastatic and metastatic organ sites are modified by extracellular vesicles and

host-derived cells, which provide an inflammatory and immune suppressive microenvironment

that is permissive for metastatic spread (24). It is not clear whether this inflammatory

microenvironment at distant sites mediates epigenetic alterations and facilitates metastatic

colonization and outgrowth. IL-6 and PGE2 are well-known pro-inflammatory factors in tumor

progression and strongly suppress host-anti-tumor immunity (25,26). IL-6 and PGE2 are critical

in premetastatic niche formation through activation of lymphatic endothelial cells and functional

alteration of dendritic cells thus promote metastasis (27,28). The IL-6/JAK/STAT3 pathway is

hyperactivated in many types of cancer and is generally associated with poor prognosis (25).

Recently, the cross-talk between IL-6 and PD1/PD-L1 was observed in the tumor




6

microenvironment and may constitute a rational immunosuppressive target for overcoming the

narrow therapeutic window of anti-PD-1/PD-L1 therapy (29).

In the current study, we demonstrate that IL-6 and PGE2 induce DNMT3B in metastatic

cancer cells at the distant sites. DNMT3B alters methylation and transcription of genes and

pathways that are important in cancer cell survival, apoptosis, proliferation, invasion, and

colonization including STAT3, NFκB, PI3K/Akt, β-catenin, as well as Notch signaling. Human

data sets also showed the increased DNMT3B levels in the distant metastases. Targeting

DNMT3B induction, in combination with chemo- or PD1- therapy, improves the treatment

efficacy in preclinical mouse models. Our study provides proof of concept and molecular

mechanisms for DNMT3B induction and its mediated epigenetic reprogramming by the

inflammatory microenvironment at the distant metastatic site. We anticipate the mechanistic

insights and preclinical evidence from the current study will aid rational design and clinical

development of combination treatment with chemo- or immunotherapy for the patients with

metastatic disease.




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Materials and Methods

Cell lines and mice. Murine mammary tumor cell lines 4T1, GFP+-4T1, EMT6, and 6DT1 were

maintained in 10% FBS DMEM (Life Technologies) and in 37 °C incubator with 5% CO2. All

cell lines are from ATCC and are mycoplasma negative with MycoAlert Mycoplasma Detection

Kit (Lonza). The cells were used within two months from thawing. Authentication has not been

performed. All mice were housed at the National Cancer Institute (NCI) animal facility, and

experiments were approved by the NCI Animal Care and Use Committee.

Plasmid constructs, transduction, and transfection. For DNMT3B knockdown or overexpression,

a psiLv-U6 empty vector or vector encoding a DNMT3B shRNA (GeneCopoeia) were stably

transduced into 4T1 and EMT6 cells. ORF-cDNA of mouse Dnmt3b (GeneCopoeia) were cloned

into the pLenti4/TO/V5-Dest vector with the Tet-inducible system using Gateway technology

(Thermo Fisher Scientific) and was stably transduced into 4T1 cells. DNMT3B expression was

induced by doxycycline for cells in vitro (1 µg/mL for 72 h) and for mice in vivo (1 mg/mL in

water). In addition, active Stat3 (Stat3-C), active -Catenin, or Notch1 intracellular domain, and

active Akt1 expressing vectors were transfected into 4T1-DNMT3B KD or control cells by

Lipofectamine®

LTX system (Thermo Fisher Scientific).

Mouse models of tumor metastasis and treatment. Eight-week-old female BALB/c, nude, or

FVB/N mice (Charles River) received 2.5-5 x 105 tumor cell injection into the mammary fat pad

(MFP) #2, or tail vein (TVI). In some experiments, TVI was performed in mice that received

MFP injection 2 weeks earlier. Mice were sacrificed on day 28-35, and the size/weight of

primary tumors or the recurrent tumors, as well as the number of metastatic nodules were




8

evaluated. For MMTV-PyMT model, the primary tumors and lung metastasis nodules were

examined from 9-month-old female mice.

For IL-6 or PGE2 effect on metastasis at distant organ site, mice received wt or

DNMT3B KD 4T1 TVI. Recombinant murine IL-6 (2.5 µg/kg bodyweight, Peprotech) or/and

16,16-dimethyl PGE2 (2.5 mg/kg bodyweight, Cayman) were i.p. injected daily.

For drug treatment, the 4T1 primary tumors were resected and the mice were treated with

Doxorubicin (2 mg/kg bodyweight, i.v. once a week), with Meloxicam (2 mg/kg bodyweight, i.p.

daily) or Etodolac (10 mg/kg bodyweight, i.p. daily). For immunotherapy, the mice were i.p.

injected with a PD-1 (5 mg/kg, BE0146) or IL-6 neutralizing antibody (10 mg/kg, BE0046) (Bio

X Cell) or the two in combination. Tumor phenotype was evaluated as indicated above. RNA

and DNA were extracted from the GFP+ metastatic nodules for molecular characterization.

Tumor cell sorting from primary tumor tissues and lung metastasis nodules. The GFP+ tumor

cells from single cell suspensions were sorted by FACS and were subjected to total RNA and

genomic DNA (gDNA) extraction.

Image Stream for DNMT3B expression levels in single tumor cells. Sorted single cells were fixed

in 4% paraformaldehyde, and incubated with DNMT3B (1:100, Abcam), and the fluorescence

intensity of DNMT3B was measured with Amnis® ImageStream MkII (Luminex Corporation).

The images were generated and analyzed by INSPIRE software (Luminex Corporation).

DNMT3B ChIP-sequencing. DNMT3B ChIP (antibody ab2851, Abcam) was performed using

Magna ChIPTM

A/G kit (Millipore). The sequence (NextSeq 500 System, Illumina) was aligned




9

on the mouse reference genome (mm10) by bowtie2 v2.1.0 and peaks were detected with MACS

v1.4 (Narrow peaks) and SICER (broad peaks), with the p-value set as > 0.05 and log2 fold-

change < 1 compared to Input. Data is available in GEO database (GSE146010).

RNA sequencing. Total RNA was extracted (TRIzol, Invitrogen), the library was prepared

(TruSeq RNA kit, Illumina), and sequenced on a HiSeq 2500 System (Illumina). FastQC and

STAR v2.4.0a alignment to the mm10 reference genome were performed to generate BAM files.

The read counts were then normalized by DESeq2 v3.1 using a scaling factor method based on

median ratio. The differential expression was determined as p- > 0.05 and log2 fold-change < 1

compared to control samples. The RNA-seq data is available in the GEO database (GSE146011).

Agilent-based target-enriched bisulfite methylation sequencing. The probes capturing the top 20%

of SICER were designed (Agilent SureDesign:

https://earray.chem.agilent.com/suredesign/home.htm), with the target enrichment of gDNA

(SureSelectXT

Methyl-seq Kit, Agilent Technologies), and bisulfite conversion (EZ-DNA

Methylation-Gold Kit, Zymo Research). The libraries were sequenced by a HiSeq 2500 System

(Illumina). The differentially methylated CpG sites were determined by the cut-offs Beta-Diff >

0.1 and FDR < 0.05. The Methylation-seq data is available in the GEO database (GSE146012).

For locus-specific DNA methylation analysis, genomic DNA with bisulfite conversion

was amplified by PCR with locus-specific primers (Takara EpiTaq HS kit, Takara Bio Inc.). The

PCR products with correct size were cloned into pCR2.1 vector system (TA Cloning Kit,

Invitrogen). Sanger sequencing was performed with 10 clones for each group.



https://earray.chem.agilent.com/suredesign/home.htm)


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ChIP-PCR and RT-qPCR. DNMT3B ChIP-PCR was performed using Magana ChIPTM

A/G

procedure. RNA was extracted (RNeasy Mini Kit, Qiagen) and cDNA was synthesized (High-

Capacity cDNA Reverse Transcription Kits, Applied Biosystem), with gene expression

determined using SYBR Green based q-PCR.

Rescue experiments for DNMT3B-targeted pathways. Proliferation: Cells were harvested every

24 h and counted in triplicates using Cellometer (Nexcelom Bioscience). Apoptosis: Cells were

labeled with 7AAD and Annexin-V (BD Bioscience), and were analyzed by BD Canto II flow

cytometry (Becton Dickinson). Wound healing assay: Cells with 100% confluence were

scratched to generate wound at the center. The recovery from the wound was monitored by

IncuCyte Live cell analysis system (Essen BioScience, Inc.). Soft agar assay: Cells were plated

in 0.3% soft agar (Sigma) with 0.6% soft agar as base layer. The number of colonies > 150 m

were counted after 12 days using FluorChem HD2 system (ProteinSimple). Sphere formation in

3D Matrigel: 4T1 single cells were cultured in medium containing 5% Matrigel (Thermo Fisher

Scientific). The number and size of tumor spheres were evaluated by phase contrast EVOS

imaging system.

Western blotting. Antibodies against DNMT3B (ab79822, Abcam), DNMT1 (NB100-56519,

Novus Biologicals), Fzd2 (sc-74019), COX2 (sc-1745), HP1 (sc-28735), and -Actin (sc-69879),

as well as DNMT3A, Nos2, p-STAT3, STAT3, p-Akt, Akt, p-p65 NFκB, p65 NFκB, β-catenin,

active β-catenin, cleaved caspase-3, Notch1, NICD1, SMAD2, SMAD3, Histone-H3, p-ERK (all

from Cell Signaling Technology) were diluted at 1/500 to 1:5000. Quantification was done using

ImageJ.




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PGE2 ELISA, cytokine antibody array, Luminex multiplex cytokine array. Protein extractions

from the lungs of Celecoxib-treated mice were examined by PGE2 Parameter Assay Kit (R&D

SystemsTM

), or by cytokine protein antibody array (Raybiotech). Additionally, inflammatory

cytokine expression was measured using Luminex Mouse Cytokine 20-plex Kit (Life

Technologies) and Precellys Lysing Kit system (Bertin Corp).

DNMT3B induction by PGE2 and IL-6. The 4T1 cells were incubated for 24 h in tissue

supernatants (from lungs of naïve or tumor-bearing mice) that were pre-incubated with IL-6 or

TGF-1 neutralizing antibody (Bio X Cell). The cells were then treated with AH-6809, an EP-

receptor antagonist (Cayman Chemical), or a STAT3 inhibitor, or sometimes with NFκB or

SMAD2 knockdown. The MDSCs used in 4T1 coculture were isolated using magnetically

activated cell sorting (MACS, Miltenyi Biotec). Protein extractions from the 4T1 cells were

examined by Western blotting.

Immunofluorescence (IF), immunohistochemistry (IHC) and TUNEL assay. Frozen lung sections

from mouse models and paraffin sections from breast cancer patients (n=7) with paired

metastatic and primary tumor tissues were incubated with DNMT3B (1:100, ab2851, Abcam) or

GFP (1:200, ab13970, Abcam, mouse only) or Phospho-H3 (1:100, Cell Signaling Technology,

mouse only) antibodies overnight at 4C. Fluorescence-tagged secondary antibody and DAPI

were utilized for visualization. Images were obtained (Carl Zeiss) and DNMT3B intensities were

quantified by ImageJ. TUNEL was performed per manufactory protocol (Roche Applied

Science).




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For circulating tumor cell (CTC), blood was collected through cardiac puncture. The

GFP+-CTCs were sorted by FACS Aria II (Becton Dickinson), were spun onto slides by cytospin,

followed by immunofluorescence staining.

Human correlation of DNMT3B expression. Publicly available datasets from Weigelt (30),

Tamura (31), Haqq (32), and Wuttig (33) were used to generate DNMT3B correlation comparing

metastasis nodules with primary tumor tissues. Additionally, paired primary and metastatic

tissues from breast cancer patients, verified by expert pathologists, were stained for DNMT3B

expression and quantified by ImageJ. The sections of formalin-fixed paraffin-embedded tissues

corresponding to Hematoxylin and Eosin-stained slides were used to ensure that at least 20% of

the retrieved cells were neoplastic.

Statistics. GraphPad Prism v6.01 and R were used for graphs and statistics. Unless otherwise

indicated, data were expressed as mean SD. All data were analyzed using the Student’s t-test

for comparison of two groups or One-way ANOVA for three groups or more. Differences were

considered statistically significant when the p-value was < 0.05.




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Results

DNMT3B is increased in metastatic nodules and is critical in metastatic colonization. An

increased expression of DNMT3B, but not DNMT3A or DNMT1, was found in metastatic

nodules compared with the primary tumors from mice that received mammary fat pad (MFP)

injection of 4T1 (Fig. 1a-b), as well as EMT6 (Fig. 1c) and 6DT1 tumor cells (Fig. 1d). In

addition to these orthotopic and spontaneous metastasis models for mammary tumors, MMTV-

PyMT mice, a transgenic mammary tumor model with spontaneous metastasis also showed

increased DNMT3B in metastatic nodules (Fig. 1e). Importantly, knockdown (KD) of DNMT3B

significantly decreased lung metastasis (Fig. 1f, 4T1 model; Fig. 1g, EMT6 model), whereas

DNMT3B induction using a tetracycline (Tet)-controlled transcriptional activation increased

metastasis in mice bearing 4T1 tumors (Fig. 1h). Taken together, these data support the critical

roles of DNMT3B in breast cancer metastasis.

The increased DNMT3B in metastatic nodules could derive from the high DNMT3B

subclonal cells in the primary tumor tissues, or as a result of DNMT3B induction at the distant

sites. The genomic and phenotypic progression outside the primary tumor is a void area of study

lacking experimental evidence, which implies new challenges and opportunities for diagnosis

and therapies aimed at metastasis. We thus chose to investigate this possibility that the increase

in DNMT3B arose outside the primary tumor. First, subclonal cell lines expressing high or low

levels of DNMT3B were established from single cell culture for both 4T1 (4T1-DNMT3BH or

4T1-DNMT3BL) and EMT6 (EMT6-DNMT3B

H or EMT6-DNMT3B

L) tumor cells (Fig. 1i,

upper panel and Supplementary Fig. 1a). Tail vein injection (TVI) of the DNMT3BH tumor cells

produced more and larger metastatic colonies compared with those from DNMT3BL tumor cells

(Supplementary Fig. 1b, 4T1; 1c, EMT6). DNMT3BH and DNMT3B

L clonal cells showed




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increased DNMT3B levels in the metastatic nodules in comparison to in vtiro culture cells (Fig.

1i, lower panels). Surprisingly, the metastatic nodules derived from DNMT3BL tumor cells

showed high DNMT3B levels comparable to those derived from DNMT3BH clones, in both 4T1

and EMT6 models (Fig. 1i, lower panels and Supplementary Fig. 1d). These data indicate the

possibility that DNMT3BL tumor cells may acquire high DNMT3B expression after entering

circulation.

Time course showing DNMT3B induction at the distant sites. To investigate the time and

location of DNMT3B induction, circulating tumor cells (CTCs) were sorted from blood

(Supplementary Fig. 1e, FACS gating) at different time points after MFP injection of GFP+ 4T1

cells (Fig. 2a, upper panel). The CTCs over 2–4 weeks showed no clear increase in DNMT3B

expression, while the tumor cells from metastatic nodules showed an increased DNMT3B (Fig.

2a, lower panels). These data again suggest DNMT3B induction at the distant sites. Next, the

GFP+ 4T1 cells were injected through the tail vein of mice pre-conditioned with MFP injection

of the GFP- 4T1 cells (without GFP). The DNMT3B levels in the GFP

+ 4T1 cells from the lungs

at different time points were measured by image stream (Fig. 2b, upper panel). DNMT3B was

significantly increased 72 h after TVI, and with the highest increase of DNMT3B in macro

metastases (>100 cells per foci) at 3 weeks (Fig. 2b, lower left panels). This result was also

observed in EMT6 mouse model (Fig. 2b, lower right panels).

To further investigate roles of the inflammatory microenvironment at the distant sites in

the DNMT3B induction, a TVI of 5 x 104 GFP+-DNMT3BL 4T1 cells (the number of cells that

did not induce an inflammatory lung microenvironment, Supplementary Fig. 1f) was performed

in mice with or without a premetastatic niche (mice bearing MFP tumors for 12–14 days) (Fig.




15

2c, schematic, left panels). There were significantly more GFP+ metastatic nodules in the mice

with the premetastatic niche than in the mice that only received a TVI of tumor cells

(Supplementary Fig. 1g). The number of GFP+ cells with high DNMT3B level was significantly

increased over time in the mice with the premetastatic niches but not in the mice without the

premetastatic niche (Fig. 2c), demonstrating the effect of inflammatory microenvironment on

DNMT3B induction. This environmental induction of DNMT3B was further supported by the

clonal cell lines derived from single cells of metastatic nodules which showed higher DNMT3B

expression compared with those from primary tumors. This difference was diminished after

several passages of in vitro culture (Fig. 2d, middle panel). However, the difference was restored

upon the treatment of the conditioned media from the lungs of tumor-bearing mice (Fig. 2d, right

panel). Together our data suggest that DNMT3B induction in metastatic cancer cells occurred at

the distant sites and resulted from tumor-associated inflammation.

Alterations in gene methylation and transcription mediated by DNMT3B in metastatic

cancer cells that bypass the primary tumor. We next investigated changes in methylation and

gene expression mediated by DNMT3B by comparing the GFP+ 4T1 cells sorted from metastatic

nodules (TVI-mets) with those sorted from primary tumors (Tumor) or spontaneous metastatic

nodules (MFP-mets) (Fig. 3a). We performed: (i) DNMT3B ChIP-seq on sorted cells from

metastatic nodules as they express high DNMT3B (Supplementary Fig. 2a for quality control);

(ii) targeted methylation-seq of sorted GFP+ 4T1 cells from TVI-mets, MFP-mets, primary

tumors, as well as cultured 4T1 cells. The capture probe design was based on genes with top 20%

DNMT3B enrichment (Supplementary Fig. 2b for quality control); (iii) RNA-seq of the above

samples (Supplementary Fig. 2c for quality control). The intersection of these datasets allows the




16

identification of differentially methylated and expressed genes targeted by DNMT3B comparing

TVI-mets, MFP-mets, and primary tumor tissues (Fig. 3b).

For DNMT3B-mediated DNA methylation changes at the distant sites comparing TVI-

mets with primary tumors, the differentially methylated CpG sites (β value difference > 0.1 and

FDR < 0.05) were detected throughout the genome, including promoter/exon 1, rest exons,

introns, 3’untranslated region (UTR), and intergenic regions (Fig. 3c). RNA-seq analysis showed

differentially expressed genes (absolute 1.5-fold changes and p < 0.05) (Fig. 3d). There was a

negative correlation between CpG methylation in promoter/exon 1 and gene expression as

exampled by Inpp5d, Cldn3, and Lfng, which are illustrated with a Janus plot that shows

hypermethylated CpG sites (-log10FDR), their genomic positions, and the changes of gene

expression (log2FC) (Fig. 3e). The promoter hypermethylation of tumor suppressors such as Lfng,

Cldn3, and Inpp5d was evident at the DNMT3B enriched regions (Fig. 3f, upper panel).

DNMT3B KD in 4T1 cells decreased DNA methylation in these regions (Fig. 3f, upper panel).

On the other hand, increased gene body methylation indicated an elevated gene expression, such

as Jag2 and Nos2 (Fig. 3f, lower panels). Of the genes identified as differentially methylated,

148 showed a profile of both differential methylation and expression comparing TVI-mets with

primary tumors (Fig. 3g, upper panel). Together, these data suggest that DNMT3B induction at

the distant sites altered the methylation and expression profile of many genes.

Between TVI-mets and MFP-mets, there were similarities and differences in methylation

(Fig. 3c, f, and Supplementary Fig. 3a) and gene expression (Fig. 3d). Of the 148 genes that were

differentially methylated and expressed between TVI-mets and primary tumors, 51 are similar

and 97 are different when compared to the MFP-mets (Fig. 3g, upper panel). Tumor suppressors,

such as Cdh11 and Tnfaip3, showed hypermethylation in the promoter region, and oncogenes,




17

such as Fzd2 and Ksr1, showed prominent hypermethylation in the gene body (Supplementary

Fig. 3b). These alterations in DNA methylation were a DNMT3B-specific effect because

DNMT3B KD decreased the methylation level (Supplementary Fig. 3b). These data support the

hypothesis that DNMT3B alters DNA methylation and gene expression in metastatic cancer cells.

DNMT3B-targeted key mediators and pathways. Ingenuity Pathway Analysis (IPA) identified

DNMT3B-targeted signaling pathways (Supplementary Table 1). The top ranked are STAT3,

NFκB, integrin-linked kinase (ILK), growth factor-receptor tyrosine kinase (RTK) pathways, β-

catenin, and Notch, as well as cell junction and extracellular matrix (ECM) remodeling (Fig. 4a),

which are important in tumor cell survival, proliferation, invasion, and colonization. Several

molecules were selected as a readout for these pathways, including pSTAT3, pNFκB, pAkt, β-

catenin, and notch1 intracellular domain (NICD-1) (Fig. 4a, upper panel red boxed). Increased

activation of these pathways were observed in both TVI-mets and MFP-mets when compared

with primary tumors and cultured 4T1 cells (Fig. 4b). Interestingly, TVI-mets and MFP-mets

shared common mediators (Fig. 4a, lower panel in red), but there were also mediators unique in

TVI-mets (Fig. 4a, lower panel in blue) or in MFP-mets (Fig. 4a, lower panel in black). These

results show while different molecular mediators could be targeted by DNMT3B comparing

TVI-met with MFP-met, they resulted the activation of the same signaling pathways. For

example, while the β-catenin pathway was activated in both TVI-mets and MFP-mets (Fig. 4b),

the Wnt ligands (Wnt 6, 7a, 9a, 10a) and Wnt sequestration molecule SFRP1, co-receptors, and

non-canonical pathway activators (Kremen1, Celsr1) were differentially expressed between TVI-

mets and MFP-mets. DNMT3B KD in 4T1 tumor cells showed a decreased activation of these

pathways in a time course experiment (Fig. 4c). Furthermore, DNMT3B KD decreased




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DNMT3B ChIP peak enrichment for the DNMT3B-targeted genes (Fig. 4d), which is consistent

with altered gene expression (Fig. 4e). Notably, DNMT3B-targeted both tumor suppressors and

oncogenes. For example, tumor suppressor Tnfaip3 which showed promoter hypermethylation,

DNMT3B KD decreased peak enrichment (Fig. 4d) and increased Tnfaip3 expression (Fig. 4e).

For oncogene Nos2 which showed gene body hypermethylation, DNMT3B KD decreased peak

enrichment (Fig. 4d) and decreased Nos2 expression (Fig. 4e). These results indicate that

DNMT3B-mediated epigenetic reprogramming and pathway activation could be achieved

through different mediators that are microenvironment-dependent. The distant organ

microenvironment contributes to this epigenetic reprogramming.

Mechanisms and biological function of DNMT3B-targeted pathways. To investigate the

molecular mechanisms and biological functions of DNMT3B mediated epigenetic

reprogramming, rescue experiments were performed in DNMT3B KD cells using constitutively

active constructs for the key signaling pathways identified above, NICD1 for Notch signaling,

myrAkt for Akt signaling, STAT3C for STAT3 signaling, and -catenin overexpression for -

catenin signaling pathways. First, in a 3D-Matrigel assay that examines the key capacity of

tumor cell colonization, re-activation of Akt, STAT3, and Notch but not -catenin signaling

pathways rescued the defect of sphere formation in DNMT3B KD 4T1 cells (Fig. 5a).

These pathways were further investigated in specific assays for colony formation,

survival, proliferation, and migration. Overexpression of the NICD1, an active form of Notch,

recovered colonization capacity of DNMT3B KD 4T1 cells in soft agar assays (Fig. 5b). Akt

activation by a constitutively active construct myrAkt rescued the defect in proliferation but not

the survival of DNMT3B KD 4T1 cells (Fig. 5c left panel and Supplementary Fig. 4a). This is




19

consistent with the observation that DNMT3B KD in 4T1 cells not only decreased the number of

lung nodules but also the size of the nodules (Supplementary Fig. 4b), tumor growth in mice

(Supplementary Fig. 4c), and decreased phospho-histone H3, a proliferative marker (Fig. 5c,

right panels). Activation of the STAT3 signaling pathway through overexpression of STAT3C

increased cell survival, which showed a decreased caspase 3 cleavage and decreased 7AAD-

/Annexin V+ population in DNMT3B KD 4T1 cells (Fig. 5d, left and middle panels). Indeed, in

metastatic nodules, TUNEL assay revealed an increased number of TUNEL+

cells in DNMT3B

KD (Fig. 5d, right panels). These observations were further confirmed in the EMT6 mammary

tumor model (Supplementary Fig. 4d-f). Furthermore, overexpression of -catenin reversed the

defect in migration and wound healing in DNMT3B KD 4T1 cells (Fig. 5e). Altogether these

results demonstrate the key mechanisms downstream of DNMT3B in epigenetic reprogramming

of metastatic cancer cells.

Mechanisms of DNMT3B induction at the distant metastatic site. To identify key

inflammatory molecules at the distant sites that are critical in DNMT3B induction, the levels of

inflammatory molecules in the premetastatic lungs of tumor-bearing mice were compared to

those in the lungs of naïve mice. PGE2 and IL-6 were the highest increased inflammatory

molecules in the premetastatic lungs (Fig. 6a and Supplementary Fig. 5a). PGE2 and IL-6 were

also detected in high levels at the late stage of metastatic lungs (Supplementary Fig. 5b).

Interestingly, the DNMT3B promoter (MatInspector, Genomatix Software) has several binding

sites for NFκB and STAT3 (Supplementary Fig. 5c) that are well-known as downstream

transcription factors of PGE2 and IL-6, respectively. Interestingly, DNMT3B was induced in

cultured 4T1 cells by conditioned medium from Day 14 and Day 35 lungs of tumor-bearing




20

mice, but not by the conditioned medium from Day 35 tumors (Fig. 6b and Supplementary Fig.

5d). The lung supernatant-induced DNMT3B was diminished by PGE2 receptor antagonist (AH-

6809) or by NFκB-p65 knockdown (Fig. 6b). Similarly, IL-6 neutralizing antibody or STAT3

inhibitor (Stattic) also inhibited the DNMT3B induction by the conditioned medium from Day

35 lungs (Fig. 6b). In contrast, neutralization of TGF- or knockdown Smad 2 did not alter the

DNMT3B induction (Fig. 6b). Furthermore, the addition of PGE2 and IL-6 to the tumor cells

cultured in vitro confirmed the induction of DNMT3B mRNA and protein level in 4T1 cells

through PGE2/NFκB (Supplementary Fig. 5e) and IL-6/STAT3 signaling (Supplementary Fig.

5f). PGE2 and IL-6 also increased DNMT3B protein levels in human breast cancer cell lines

(Supplementary Fig. 5g).

The inflammatory premetastatic and metastatic lung is modulated by myeloid-derived

immune suppressor cells (MDSCs or Gr-1+CD11b

+ cells) that are well known to suppress host

immune surveillance (34,35). In an in vitro coculture of tumor cells with MDSCs, MDSCs

stimulated DNMT3B induction in tumor cells (Fig. 6c). PGE2 receptor antagonist (AH-6809)

and IL-6 neutralizing antibody diminished DNMT3B induction and consistently inhibited NFκB

and STAT3 signaling (Fig. 6c). These data suggest DNMT3B induction is mediated by MDSCs

through PGE2 and IL-6.

Because of the high PGE2 concentration in metastatic lungs, Celecoxib, a COX2

inhibitor, was used to decrease PGE2 and suppress the inflammation in the metastatic

microenvironment. Celecoxib treatment significantly decreased PGE2 levels in the lungs of

tumor-bearing mice (Fig. 6d) and inhibited DNMT3B induction in lung nodules of both 4T1 and

EMT6 models (Fig. 6e). To confirm the epigenetic regulation of DNMT3B targeted genes by

inflammation in vivo, Cldn9 and Lfng, negative regulators of β-catenin and Notch pathways




21

respectively, were selected as two representative DNMT3B-targeted molecules because of strong

DNMT3B peak enrichment and hypermethylation in their promoters (Supplementary Fig. 5h).

Celecoxib treatment decreased CpG site methylation and increased gene expression of Cldn9 and

Lfng in metastatic lung nodules (Fig. 6f). Importantly, overexpression of DNMT3B using Tet-

inducible system reversed the effect of Celecoxib on DNA methylation and gene expression of

Clnd9 and Lfng (Fig. 6f). Lastly, treatment with IL-6 or PGE2 or the two in combination

increased the number of lung metastatic nodules in mice that received tail vein injection of wt

but not the DNMT3B KD 4T1 tumor cells, suggesting the effect of these inflammatory mediators

on metastasis at the distant site is DNMT3B dependent (Fig. 6g). Altogether these data further

demonstrate that DNMT3B is induced in the distant metastatic site through PGE2/NFκB and IL-

6/STAT3 signaling pathways. DNMT3B alters gene methylation and transcriptome leading to

the activation of signaling pathways that are important in metastatic colonization (Fig. 6h).

Correlation of DNMT3B with human tumor metastasis, perioperative COX2 inhibition in

combination with chemotherapy, and IL-6 neutralization in combination with PD-1

immunotherapy in preclinical mouse models. We next used publicly available clinical datasets

and correlated DNMT3B expression levels with metastatic progression. DNMT3B but not

DNMT1 and DNMT3A was increased in metastatic samples compared with matched primary

tumors of breast cancer patients (Fig. 7a and Supplementary Fig. 6a) (30). This result was also

observed in prostate cancer patients (Fig. 7b, and Supplementary Fig. 6b) (31), in melanoma

patients (Fig. 7c, and Supplementary Fig. 6c) (32) as well as in renal carcinoma patients (Fig.7d,

and supplementary Fig. 6d) (33). In 7 cases of breast tumor samples obtained, distant metastatic

tumors showed significantly more cells with elevated DNMT3B than that from the matched




22

primary tumors (Fig. 7e). These data suggest a positive correlation of DNMT3B expression

levels with metastatic progression.

Increased PGE2 levels in the circulation were observed in cancer patients after tumor

resection surgery, and the systemic inflammation by the COX2/PGE2 pathway was suggested to

facilitate early relapse and metastasis (36). Our study showed that PGE2 was the most

significantly increased inflammatory mediator in the pre-metastatic sites (Fig. 6a), and PGE2

induced DNMT3B in breast cancer cells (Fig. 6b, and Supplementary Fig. 5e). Therefore, we

investigated the effect of perioperative inhibition of the COX2/PGE2 pathway using COX2

inhibitors, Meloxicam (Mel) or Etodolac (Eto), in 4T1 preclinical mouse model (Fig. 7f

schematic design). The perioperative inhibition of COX2 significantly reduced the number of

lung nodules, and the treatment of COX2 inhibitors further enhanced anti-metastasis efficacy of

Doxorubicin (Dxr) chemotherapy (Fig. 7g, left panel, and Supplementary Fig. 6e). Treatment of

Doxorubicin alone or co-treatment of Doxorubicin with COX2 inhibitors significantly

suppressed recurrent tumor growth (Fig. 7g, right panel).

DNMT3B is also induced by IL-6 (Fig. 6b and c). The IL-6/JAK/STAT3 signaling is

known to suppress the antitumor immune response. In addition, agents targeting IL-6 signaling

pathways have already received FDA approval for the treatment of inflammatory conditions

(25,37). Therefore, we next explored whether IL-6 neutralization will improve the efficacy of

PD-1 immunotherapy that has a low response rate but acceptable safety profile and antitumor

activity in patients with TNBC. In the preclinic 4T1 mouse model (Fig. 7f, schematic design),

IL-6 neutralization or anti-PD-1 immunotherapy alone decreased the number of metastatic

nodules. Importantly, IL-6 neutralization potentiated the attenuation of lung metastasis by PD-1

neutralization Ab (Fig. 7h, left panel, and supplementary Fig. 6f). Consistently, the number of




23

metastasis-free mice was markedly increased in mice that received the combination treatment

(Fig. 7h, middle panel). IL-6 neutralization also enhanced the attenuated recurrent tumor growth

by PD-1 neutralization (Fig. 7h, right panel). These data suggest that IL-6 neutralization could be

used to enhance the efficacy of PD-1 immunotherapy. Altogether the enhanced PD-1

immunotherapy by IL-6 neutralization or Doxorubicin efficacy by Meloxicam is striking and

clear, with no need to combine COX-2 inhibition with IL-6 neutralization for the immunotherapy

or Doxorubicin treatment. This is consistent with the observation that the combination of

Meloxicam and IL-6 neutralization showed no significant decrease in metastasis, recurrent

tumor growth, or number of CTCs when compared with that of the single treatment alone

(Supplementary Fig. 7a, b, and c).




24

Discussion

Our study provides experimental evidence and conceptual proof that DNMT3B is induced at the

metastatic site and mediates epigenetic reprogramming for metastatic colonization. Our results

offer insight into metastasis biology and accentuate the inherent insufficiency and daunting

challenge of current treatment strategies. Therapies based on the genetic characterization of

primary tumor biopsies can only prolong the lives of patients with metastatic disease by a few

weeks or months (1,38). While metastatic driver mutations are continually investigated and

identified, the current study asserts that epigenetic acquisition of malignant traits after tumor cell

dissemination must be taken into consideration. Our study also suggests that epigenetic

regulation is likely critical in manifestation of full malignancy for early disseminated cancer cells

(39), especially in the absence of driver mutations.

Our work reveals a new role of inflammation in DNMT3B induction and bolsters

evidence that there is a pronounced link existing between inflammatory pathways and epigenetic

mechanisms. Interestingly, NFκB and STAT3 pathways are not only upstream of the DNMT3B

induction but are also activated as DNMT3B downstream effectors. This potential feedback loop

indicates an epigenetic mechanism to mediate transcriptional reprogramming of cancer cells

which could facilitate metastasis in less microenvironment dependent manner. A recent study

showed transcriptional reprogramming of resistant signatures was acquired in response to

chemotherapy in TNBC patients (40), suggesting an adaptation mechanism under stress

conditions. Our data suggest the metastatic microenvironment modifies tumor cells

epigenetically thus reprogramming the transcriptome. Cancer-associated inflammation is critical

in tumor progression. However, whether and how the cancer-associated inflammation affects

metastatic colonization at distant sites remains unclear. IL-6 and PGE2 are two potent pro-




25

inflammatory mediators known to drive the production, accumulation, and immune suppressive

potency of MDSCs (41). We now show that premetastatic/metastatic lungs produced high levels

of IL-6 and PGE2. Additionally, IL-6 and PGE2 play an important role in DNMT3B induction in

metastatic cancer cells at the distant sites. Furthermore, DNMT3B was induced in cancer cells

when co-cultured with MDSCs that were abundant in the premetastatic and metastatic distant

organ microenvironment (35). A recent study of DNA methylation landscape reported the

significant correlation between DNA methylation and immune cell infiltration in primary and

recurring human glioblastomas, providing the clinical evidence for the link between tumor

epigenome and immune cells (42). Further investigation utilizing genetically engineered mouse

models to manipulate specific host immune cells will provide more insight about epigenetic

regulation of tumor cells by microenvironment in vivo. Nevertheless, our data suggest that the

inflammatory microenvironment at the distant organ sites promotes tumor metastasis through

DNMT3B mediated epigenetic mechanism.

Our study shows that perioperative inhibition of COX2/PGE2 pathway using Meloxicam

or Etodolac reduced the number of metastatic nodules in 4T1 preclinical mouse model.

Importantly, it significantly enhanced anti-metastasis efficacy of Doxorubicin (Fig. 7f, g, left

panel, and Supplementary Fig. 6e). Further, there was also profound effect on recurrent tumor

growth when used in combination with Doxorubicin (Fig. 7f, g, right panel). There has been

increased understanding that tumor resection induces systemic inflammation which mobilizes

inflammatory immune cells and promotes tumor relapse (43). In fact, surgical removal of

primary tumors in ER-positive breast cancer patients correlated with increased tumor recurrence

and metastases 1-2 years post tumor resection (44). In addition, neoadjuvant chemotherapies

increase secretion of pro-metastatic extracellular vesicles and to induce the formation of pro-




26

metastatic microenvironment in preclinical mouse models of breast cancer (45,46). The

significantly decreased metastasis by the short-term perioperative treatment of COX2 inhibitors

in combination with Doxorubicin neoadjuvant chemotherapy suggests a unique therapeutic

window which can provide long-term benefit for cancer patients. Moreover, the usage of

common and inexpensive anti-inflammatory drugs provides additional value to this therapeutic

approach.

In our study, IL-6 neutralization enhanced the efficacy of PD-1 immunotherapy for breast

cancer metastasis in the preclinical mouse models. Significant evidence supports immunotherapy

for breast cancer treatment (47). A recent clinical trial demonstrated that co-treatment of PD-L1

antibody (Atezolizumab) and Nab-paclitaxel prolonged progression-free survival in patients with

advanced triple-negative breast cancer (3), leading to the first FDA approval of immunotherapy

in breast cancer. However, the complete response rate was only 7.1% in patients who received

the co-treatment (3). Indeed, low response rate, therapy relapse and resistance are major

challenges in immunotherapy (2). The IL-6/JAK/STAT3 pathway could contribute to these

challenges, because this pathway strongly suppresses host-anti-tumor immunity and generally

correlates with poor prognosis (25). In fact, PD-1 blockade elevates IL-6 mediated inflammatory

response in macrophages, and IL-6 inhibition alleviates this adverse effect (29). The crosstalk

between IL-6 and PD1/PD-L1 could be utilized to overcome the narrow therapeutic window of

anti-PD-1/PD-L1 therapy. These studies and our data support our proposal that IL-6 blockade

could provide an important option to enhance the efficacy of PD-1 immunotherapy for metastatic

TNBC patients. We anticipate IL-6 neutralization will directly inhibit the epigenetic

dysregulation in tumor cells and at the same time enhance host anti-tumor immunity.




27

It is not fully understood how DNA methylation is regulated, and the transcriptional

outcome of DNA methylation seems to vary with genomic context (48). Our data support a

promoter/exon 1 hypermethylation in down regulation of many tumor suppressor genes such as

apoptosis mediators (e.g., TNFAIP3), cell adhesion molecules (e.g., CDH11), and phosphatases

(e.g., Ptpn7, Inpp5d). Conversely, DNA hypermethylation in the gene body generally positively

correlates with increased oncogene expression, such as Nos2, Fzd2, Jag2. These results are

consistent with other epigenetic studies (17,18,22). Interestingly, we notice that the DNMT3B

ChIP-seq peaks do not always result in alterations of DNA methylation. Among DNMT3B-

enriched regions, 22.0% showed changes in CpG methylations. Of particular note, DNMT3B

does not possess a specific DNA binding domain. Rather it has a PWWP domain that interacts

with histone protein markers (49). We suspect that specific histone modifications may mediate

DNMT3B recruitment in certain gene loci. Indeed DNMT3B is recruited to H3K36m3 for gene

body methylation in mouse embryonic stem cells and is critical in transcription fidelity in

controlling splicing variants (50). A recent report also shows that CpG islands are pre-marked by

H3K4me1 in normal prostate cells but are susceptible to DNMT3B mediated hypermethylation

at the boundaries of those CpG islands in prostate cancer cells (51). Further studies of histone

modifications that are specific to metastatic tumors will provide more comprehensive

understanding of epigenetic regulation in metastatic progression.

Unsurprisingly, but importantly, DNMT3B targets not only multiple genes in the same

pathway, including positive and negative mediators, but also multiple pathways (Fig. 4). This

global epigenetic reprogramming of metastatic cancer cells is also reported for small cell lung

cancer cells that acquire chromatin accessibility at distal regulatory regions and facilitate the

expression of pro-metastatic genes (52). Because of the magnitude of DNMT3B-mediated




28

alterations, our work argue that: 1) the current targeting strategy based only on genomic

alterations with combination of 2–3 drugs are unlikely to be sufficient; 2) DNMT3B-mediated

epigenetic alterations must be considered in therapeutic design; 3) a feasible anti-inflammatory

approach that targets DNMT3B induction and enzymatic activities will likely restore tumor

suppressor genes and improve efficacy of standard therapy. This treatment approach may also be

useful in addressing therapeutic resistance mediated by the tumor microenvironment and its

poised epigenetic states (53). The mechanistic insights from our study have implications

regarding the steps of the metastatic adaptation and colonization that appear amenable to

therapeutic targeting and metastasis prevention.




29

Acknowledgements

We thank Dr. Michael Bustin for his critical reading of the manuscript. We thank Dr. Hua Yu

(City of Hope) for providing the Stat3-C vector, and Dr. Lalage Wakefield (NCI) for providing

shRNA for SMAD2 and SMAD3. We appreciate the CCR Flow Cytometry Core for their

technical assistance on FACS. We would like to thank Cindy Clark, NIH Library Writing Center,

for manuscript editing assistance.

The studies are supported by the US government (NCI) intramural funding to Dr. Li Yang




30

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Figure Legend

Fig. 1 Induction of DNMT3B and Its Effects in Metastatic Colonization. a DNMT3B, DNMT3A,

and DNMT1 Western, protein extraction from 4T1 primary tumors and lung nodules. b

DNMT3B IHC of 4T1 lung nodules and primary tumors. c & d DNMT3B, DNMT3A and

DNMT1 Western of primary tumors and lung nodules from EMT6 (c) and 6DT1 (d) orthotopic

mammary tumor models. e DNMT3B Western of primary tumors and lung nodules for MMTV-

PyMT transgenic mice. f & g The effect of DNMT3B KD on metastasis in 4T1 (f) or EMT6 (g)

experimental metastasis models. Western for DNMT3B KD (upper left panels), and metastatic

nodule counts from mice injected with DNMT3B KD (n=8) or control cells (n=8) (right panels).

Representative lung images are shown in lower left panels. h Western for DNMT3B

overexpression by doxycycline (Dox) in Dox-inducible DNMT3B expression in 4T1 cells (upper

panel), and metastatic nodule counts from mice bearing 4T1 tumors overexpressing DNMT3B

(n=6) (lower panel). i DNMT3B Western for subclonal 4T1 and EMT6 cell lines with high or

low DNMT3B expression. For Fig. a, c, d, i: each lane is from an individual mouse. The

quantification of DNMT3B expression by band density is listed for each blot as shown, and

the values were normalized to -Actin. Data are presented as mean ± standard deviation (SD). *p

< 0.05, **p < 0.01.

Fig. 2 Time Course of DNMT3B Induction at the Distant Sites. a Schematic experimental design

(upper panel) and DNMT3B immunofluorescence (IF) staining in sorted CTCs at different time

points, and sorted tumor cells from 4T1 metastatic nodules as positive control. Representative

photos and quantitated DNMT3B levels (lower panel). b Schematic experimental design for TVI

in mice with premetastatic niche (upper panel); Image stream of DNMT3B expression levels in




35

TVI-injected GFP+ 4T1 and GFP

+ EMT6 cells from the lungs at different time points.

Representative photos and quantitated DNMT3B levels of GFP+ 4T1 cells (lower left panel) and

GFP+ EMT6 cells (lower right panel). c DNMT3B IF staining of metastatic loci at different time

points after TVI of DNMT3BL GFP

+ 4T1 cells in mice with or without premetastatic niche:

schematic experimental design (left panel); representative images (middle panels); number of

GFP+ 4T1 cells with high DNMT3B expression (n=6 sections for each sample, right panel). d

DNMT3B expression of in vitro clonal cell culture over passages: schematic experimental

design (left panel); RT-qPCR of DNMT3B expression in clonal cell culture from metastatic

nodules compared to those from primary tumor and 4T1 in culture (middle panel); RT-qPCR of

DNMT3B expression in clonal cell culture treated with or without D35 lung extract from mice

bearing 4T1 MFP tumors (right panel). Number of passages in culture and treatment conditions

were indicated. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3 DNMT3B Alters Methylation and Gene Expression in Metastatic Cancer Cells. a

Schematic for metastatic GFP+ 4T1 cells that bypass the primary tumor (upper panel), or from

MFP-mets and primary tumors (lower panel). b Analysis strategies for DNMT3B-ChIP-seq,

target-enriched bisulfite methylation-seq, and RNA-seq. c Heatmap of differentially methylated

CpG sites comparing TVI-mets with primary tumors as well as MFP-mets. Cut-off: Beta-Diff >

0.1 and FDR < 0.05. β value 1 corresponding to 100% methylation. d Heatmap of differentially

expressed genes comparing TVI-mets with primary tumors as well as MFP-mets. e Janus plot for

the differential promoter methylation and gene expression comparing TVI-mets vs primary

tumor. Example genes with increased promoter methylation and decreased expression are

highlighted with colored dots (Inpp5d in red; Cldn3 in green; Lfng in blue). The upper portion




36

(above the yellow line) of the plot shows hypermethylated CpG sites (-log10FDR), and the lower

part of the plot (below the yellow line) shows log fold change of gene expression (log2FC).

Heatmaps show methylation β values of individual CpG in the promoter regions comparing TVI-

mets with primary tumor. Bar graphs below show gene expression levels in TVI-mets and

primary tumors. f Genomic Browser view of DNA methylation and DNMT3B-ChIP for

representative genes with differential methylation and expression. Green indicates down-

regulated genes (Inpp5d, Cldn3, Lfng), orange indicates up-regulated genes (Jag2 and Nos2).

Peaks in red indicates hypermethylation and the ones in blue indicates hypomethylation

comparing TVI-met/primary tumor, MFP-mets/primary tumor, as well as DNMT3B KO vs WT.

g Upper: Venn diagrams of differentially methylated and expressed genes. Lower: Heatmap for

differentially methylated and expressed genes comparing TVI-mets with primary tumor as well

as MFP-mets. Mediators for key pathways are indicated on the right side.

Fig. 4 DNMT3B Alters Key Mediators in STAT3, NFκB, PI3K/Akt, β-catenin, as well as Notch

Signaling Pathways. a Schematic major signaling pathways and key genes targeted by DNMT3B.

Red line labeled molecules pSTAT3, pNFκB, pAKT, β-Catenin, and NICD-1 are used as

readouts for the corresponding signaling pathways (upper panel). DNMT3B-targeted pathways

and genes comparing TVI-mets and MFP-mets. Common genes between TVI-mets and MFP-

mets are in red; unique genes in TVI-mets are in blue; unique genes in MFP-mets are in black

(lower panel). b Western blots of DNMT3B-targeted genes and pathways comparing TVI-mets,

MFP-mets, primary tumors, and 4T1 cells. Each lane is from an individual mouse. c Western

blots of DNMT3B-targeted genes and pathways from DNMT3B KD or wt 4T1 cells. d

DNMT3B-ChIP-qPCR from DNMT3B KD or wt 4T1 cells. e RT-qPCR for mRNA expression




37

in DNMT3B KD over wt 4T1 cells (log2 fold changes). Red: common genes between TVI-mets

and MFP-mets; blue: unique genes in TVI-mets; black: unique genes in MFP-mets. Data are

presented as mean ± SD. *p > 0.05, **p > 0.01.

Fig. 5 DNMT3B Activates Pathways that are Important in Cancer Cell Survival, Apoptosis,

Proliferation, Invasion, and Colonization. a Tumor sphere formation in 3D Matrigel, re-

activating Akt (myAkt), STAT3 (STAT3C), and Notch (NICD-1) but not -catenin signaling

pathways rescued the defect of DNMT3B KD 4T1 cells in tumor sphere formation. Shown are

representative pictures (left) and quantitative data (right). b NICD1 Western (upper panel) and

colony formation (lower panel) of DNMT3B KD 4T1 cells with a NICD1 construct. c pAkt

Western blot (left upper panel) and cell counts (left lower panel) for myrAkt rescued

proliferation defect in DNMT3B KD 4T1 cells. Right panels: IHC of phospho-histone H3 of

lung nodules from mice injected with DNMT3B KD or wt 4T1 cells. The quantitative data are on

the right, with the number of positive cells per lung nodule from 20 nodules of comparable size.

d pSTAT3 and c-Casp3 Western (left upper panel), percentage of 7AAD-/Annexin V

+ population

(left lower panel) and representative flow cytometry (middle panels) of DNMT3B KD 4T1 cells

transfected with STAT3C. Right panels: TUNEL assay of lung nodules from mice injected with

DNMT3B KD or wt 4T1 cells. Quantitative data on right with the number of TUNEL+ cells per

lung nodule from 20 nodules of comparable size. e Wound closure assay of DNMT3B KD 4T1

cells with a constitutive active -catenin construct. Representative pictures of wound closure at

15 hr on the right. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.




38

Fig. 6 Inflammatory Mediators PGE2 and IL-6 Induce DNMT3B. a ELISA and Bioplex assays

of protein extraction from normal lungs and lungs from 4T1 tumor-bearing mice (n=3–6 lungs).

b Western blots for DNMT3B, pSTAT3, pNFκB and pSmad2 of 4T1 cells cultured with lung

tissue culture supernatants, and with neutralization of TGF-, IL-6 or/and PGE2 receptor

antagonist (AH-6809), as well as knockdown of NFκB or Smad2 or treatment with STAT3

inhibitor (Stattic). c Western blots for DNMT3B, pSTAT3, pNFκB in an in vitro coculture of

4T1 cells with MDSCs, with or without PGE2 receptor antagonist (AH-6809) and IL-6

neutralizing antibody treatment. d PGE2 ELISA of protein extraction from normal lungs and

lungs from tumor-bearing mice with or without Celecoxib treatment. e DNMT3B Western of

lung nodules of 4T1 (left panel) and EMT6 (right panel, each lane from an individual mouse)

tumor-bearing mice treated with Celecoxib, normal lung or primary tumors are used as controls.

Days after MFP tumor injection is indicated. f Locus-specific DNA methylation analysis and

RT-qPCR of DNMT3B target genes Cldn9 and Lfng. g Number of lung metastatic nodules from

mice that were treated with PGE2 or IL-6 or in combination. The DNMT3B KD or wt control

4T1 tumor cells were injected through tail vein, representative images shown on the right. h

Schematic hypothesis: MDCSs, PGE2, and IL-6 are increased in the inflammatory metastatic

microenvironment, which induce DNMT3B in the metastatic cancer cells through PGE2/NFκB

and IL-6/STAT3 signaling pathways. DNMT3B alters gene methylation and transcriptome thus

activates signaling pathways including STAT3, NFκB, PI3K/Akt, β-catenin, as well as Notch

signaling pathways that are important in metastatic colonization. Data are presented as mean ±

SD. *p < 0.05, **p < 0.01.




39

Fig. 7 Correlation of DNMT3B with Human Tumor Metastasis, Perioperative COX2 Inhibition

in Chemotherapy, and IL-6 Neutralization in PD-1 Immunotherapy in Preclinical Mouse Models.

a DNMT3B expression in paired primary and metastatic tumor tissues from breast cancer

patients (Weigelt dataset, n=8). b, c, d DNMT3B expression in primary tumor and metastases of

prostate cancer patients (b, Tamura dataset), melanoma patients (c, Haqq dataset) and renal cell

carcinoma (d, Wuttig dataset). e H&E and DNMT3B IF for matched primary and metastatic

breast tumors (n=7 cases). Representative images are shown, with quantitative data on right. f

Schematic of experimental design for perioperative anti-inflammatory therapy and IL-6

neutralization in combination with anti-PD-1 immunotherapy. Animals were treated with

Doxorubicin (Dxr, 2 mg/kg bodyweight), Meloxicam (Mel, 2 mg/kg bodyweight), Etodolac (Eto,

10 mg/kg bodyweight), IL-6 neutralizing antibody (IL-6 Ab, 10 mg/kg bodyweight), or PD-1

neutralizing antibody (PD-1 Ab, 5 mg/kg bodyweight) as shown in the design. g The effect of

perioperative anti-inflammatory therapy on 4T1 metastasis (n=10). The number of metastatic

lung nodules (left panel), and the weights of recurrent tumors (right panel). h The effect of IL-6

Ab in combination with PD-1 Ab on 4T1 metastasis (n=9-10). The number of metastatic lung

nodules (left panel), the percentage of metastasis-free mice (middle panel), and the weights of

recurrent tumors (right panel), are graphed. Data are presented as mean ± SD. *p < 0.05, **p <

0.01, ***p < 0.001.




Published OnlineFirst April 7, 2020.Cancer Res Li Yang, Jae Young So, Nicolas Skrypek, et al. colonizationsites promotes epigenetic modification and breast cancer Induction of DNMT3B by PGE2 and IL6 at distant metastatic

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